The goal of this proposal is to characterize in detail the age-associated epigenetic alterations and their functional roles in the age-related risk for cancer, so that we can devise approaches to either prevent or treat early neoplasms. This proposal builds on our current ongoing and recently published work indicating that cancers derive from ageing, dividing cells due to accumulation of epigenetic changes. Multiple studies have shown that normal ageing involves accumulation of epigenetic alterations involving DNA methylation, modifications to histone and non-histone proteins, and chromatin structure. However, most studies have focused on DNA methylation, and the functional roles of these various age-related epigenetic modifications in promoting tumorigenesis has been lacking. Our work uses novel approaches employing ex vivo colon-derived stem cell organoid and in vivo mouse models. In preliminary data, we show that ?ex vivo ageing? of mouse colon organoids involves evolution of promoter DNA hypermethylation, akin to in vivo ageing, which facilitates activation of the Wnt-pathway and predisposes to transformation by oncogenic-Braf. To obtain in-depth insights into alterations in the transcriptome due to age-associated epigenetic changes, and its impact on tumor initiation, we will map key histone modifications (H3K4me3, H3K27me3, H3K27ac), DNA methylation and chromatin structure (open/closed chromatin configuration) in colon epithelial cells from mice of different age groups. Changes to gene regulation mediated by epigenetic alterations in promoters and other genomic regulatory elements, such as enhancers, will be identified. The genomic analyses will be followed by functionally testing the roles of important age-altered genes and pathways in promoting tumor initiation using the novel organoid-based ex vivo tumorigenesis assays. Importantly, our work focuses on understanding the epigenetic changes in long-lived colon epithelial stem cells, from which tumors most likely derive. Our current data invokes the concept that epigenetic events in stem cells, arising in the context of ageing, may initially allow escape from senescence, retention of stem cell characteristics, and cause differentiation defects. We hypothesize that minor stem cell subpopulations with such characteristics exist in aged tissues and are predisposed to tumorigenesis in the context of cancer driver mutations. We will test this hypothesis using ex vivo organoids generated form mice of different age groups, and enriching for cells with increased stem cell properties/differentiation defects. Finally, we will directly test if epithelial stem cells from aged organoids are predisposed to tumorigenesis in the context of oncogenic mutations. In ongoing work we have teamed up with Dr. Rafael de Cabo (NIA); the complementary expertise of our groups in age-related disorders (de Cabo) and cancer epigenetics (Baylin, Easwaran) will help achieve the stated goals. Ultimately, our findings using the colon model will be applicable to other tumor types and to the human ageing scenario in general, which will have high impact on strategies to reduce age-related cancer risk.